The Fanconi anemia/homologous recombination (FA/HR) pathway is frequently silenced by mutations or epigenetic processes in several types of cancer, including high-grade serous ovarian cancer (HGSOC), basal breast cancer, and a subset of pancreatic and prostate cancers. As a result, high fidelity repair of DNA double- strand breaks is disabled, leading to genomic instability in these cancers. Inhibitors of the DNA repair protein poly(ADP-ribose) polymerase (PARP) have shown promising activity in HR-deficient preclinical models and response rates of 30-45% in BRCA1 or BRCA2 (BRCA1/2) mutation carriers with platinum-sensitive relapsed HGSOC. This project is designed to better understand why some BRCA1/2-mutant cancers respond to PARP inhibitors and others do not. Previous results from members of our investigative team have not only identified BRCA1/2 reversion mutations as a potential cause of platinum resistance in preclinical models and clinical samples, but also demonstrated that the cytotoxic effects of PARP inhibitors reflect activation of nonhomologous end-joining (NHEJ), an error-prone DNA double-strand break repair pathway, rather than inhibition of DNA single-strand break repair as originally thought. Importantly, inhibition or downregulation of any of a number of NHEJ proteins inhibits this error-prone repair and diminishes the cytotoxic effects of PARP inhibitors. This new understanding of PARP inhibitor action leads to the hypothesis that resistance of BRCA1/2-mutant cancers to PARP inhibitors can result from either reversion mutations or changes in DNA repair pathway proteins that abrogate the action of the PARP inhibitors. Consistent with this hypothesis, our further studies in a BRCA2-mutant preclinical ovarian cancer model have demonstrated that selection for PARP inhibitor resistance results in either downregulation of NHEJ proteins or overexpression of Rad51, an HR protein downstream of BRCA2. To build on these findings we now propose to: i) further assess the impact of NHEJ protein downregulation on PARP inhibitor sensitivity in vitro and in vivo, ii) examine how Rad51 is upregulated to induce PARP inhibitor resistance; iii) perform a whole-exome shRNA screen looking for additional mechanisms of PARP inhibitor resistance in BRCA1/2-mutant ovarian cancer lines in vitro; and iv) examine two unique sets of cancer biopsies from patients receiving single-agent PARP inhibitor therapy to evaluate the potential importance of these resistance mechanisms in the clinical setting. Collectively, these studies will provide new insight into mechanisms of PARP inhibitor resistance that will enable future selection of ovarian cancer patients most likely to benefit from these promising new drugs.